Our research plan is focused on generating a comprehensive understanding of G-protein-coupled receptors (GPCRs) and their signaling partners on epithelial stem cell proliferation and differentiation. Our previous findings indicate that GPCR regulation of cAMP signaling may be involved in differentiation and tumorigenicity within the epidermal stem cell compartment. Here we plan to characterize whether Galphai-coupled receptors are involved in this process and determine the role of YAP1 signaling in regulating cell fate downstream of GPCRs. A) Identify and define the role of Galphai signaling in epithelial cells. To understand how the balance of cAMP levels regulates cell fate in the epidermis, we are using muscarinic designer receptors (DREADDs) coupled to Galphai (hM4Di). Our results indicate that Galphai activation by hM4Di in the skin in vivo and in human keratinocytes and 3D models in vitro leads to increased keratinocyte proliferation and decreased differentiation, resulting in skin hyperplasia. Preliminary data indicates that Galphai-mediated keratinocyte proliferation and decreased differentiation might be the result of activation of YAP1. We are currently dissecting the effects of Galphai on hedgehog signaling and ciliary cAMP. In addition, we are characterizing the GPCRs expressed in keratinocytes, the function of specific GPCRs and possible pharmacological interventions that can be used to modulate keratinocyte biology. B) Identification of transcriptional circuitries regulated by YAP1/TAZ downstream of GPCRs. Galphas and Galphai signaling in the skin regulate YAP1/TAZ nuclear translocation and activation, indicating that YAP1/TAZ could mediate nuclear events downstream of GPCRs. By using a newly developed genetically-encoded inhibitor of YAP1/TAZ-mediated transcription, we are identifying the gene networks regulated by this signaling pathway in human skin by utilizing normal and cancer cell lines as well as a newly established animal model. The YAP1/TAZ inhibitor will allow us to determine the precise role of YAP1/TAZ in normal cell homeostasis and in cancer initiation and progression. In addition, we will be able to test the role of YAP1/TAZ/TEAD in transducing mitogenic signals and other transcriptional events initiated by GPCR signaling.